(a) Field of the Invention
The present invention relates to a novel fusion protein comprising Staphylococcal protein A and mussel adhesive protein, a biochip comprising a solid substrate to which the fusion protein is attached, and a method for detecting a target antigen in a biological sample using the biochip. Furthermore, the present invention relates to a polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, a transformed cell comprising the recombinant vector, and a method of preparing the fusion protein by transformed cell comprising the recombinant vector.
(b) Description of the Related Art
Early detection of diseases is a key for their successful treatments despite of advancement of therapeutic tools. Immunoassay is one of the vital technologies for disease diagnosis due to high specificity and affinity of antibodies to target antigens. In most of immunoassays, antibody-antigen interactions are measured on solid supports. Because effective immobilization of antibody on solid support controls specificity and sensitivity of assay devices, immobilization of antibody with efficient manner is crucial and one of key issues for various fields including immunoassays and immunosensors.
Antibodies have been immobilized on solid supports by diverse methods including physical adsorption and covalent binding (R. Pei, X. Yang, E. Wang, Analyst 2001, 126, 4.; A. Subramanina, J. Irudayaraj, T. Ryan, Biosens. Bioelectron. 2006, 21, 998.). However, these methods have been suffered from reduced antibody activity (that is, antigen binding ability) due to random orientation, denaturation, and chemical modification of antibodies (G. T. Hermanson, Bioconjugate Techniques, Academic press, California 1996.; H. Zhu, M. Synder, Curr. Opin. Chem. Biol. 2003, 7, 55.). Therefore, antibody-binding proteins such as protein A and G have been widely employed to overcome limitations of the previous physical and chemical methods. Those proteins specifically recognize and bind to Fc region of antibodies with high affinity, and realized oriented-immobilization of antibodies on surfaces without reduced antibody activity (M. D. P. Boyle, K. J. Reis, Biotechnology 1987, 5, 697.; W. L. Hoffman, D. J. O′ Shannessy, J. Immunol. Methods 1998, 112, 113.). Thus, the antibody-binding protein-based antibody immobilization methods have significantly improved antigen-binding ability, sensitivity, and stability compared to the previous methods.
However, efficient attachment of antibody-binding proteins on solid surfaces still remains as a major challenge. For efficient binding with functional orientation, antibody-binding proteins have been fused with several biomolecules such as cysteine residue(s), oligonucleotide, hexahistidine (His6) peptide, gold-binding peptide, and glutathione-S-transferase (J. M. Lee, et al., Anal. Chem. 2007, 79, 2680.; Y. Jung, et al., Anal. Chem. 2007, 79, 6534.; S. M. Patrie, et al., Anal. Chem. 2007, 79, 5878.; S. Ko, et al., Biosens. Bioelectron. 2009, 24, 2592.; T. H. Ha, et al., Anal. Chem. 2007, 79, 546.). However, the above fusion proteins have still limitation because immobilization is possible onto only corresponding reactive group-derived surfaces. For protein immobilization, preparation of modified surface is time-consuming and sometimes difficult for non-experts.
Mussel adhesive proteins (MAPs) from marine mussels are water-insoluble bioadhesives that adhere tightly to substrata. MAPs are able to form strong bond to diverse substrates including glass, metal, and plastics without any surface pretreatments (J. H. Waite, Int. J. Adhesion Adhesive 1987, 7, 9.; L. O. Burzio, et al., The Japanese Soc. Mar. Biotechnology., Tokyo, 1989.). However, the amounts of MAPs that can be extracted directly from mussels are extremely low and attempts to produce functional recombinant MAPs have been failed (D. Morgan, Scientist 1990, 4, 1; M. Kitamura, et al., J. Polym. Sci. Ser. A 1999, 37, 729; A. J. Salerno, et al., Appl. Microbiol. Biotechnol. 1993, 58, 209.).
In the present work, we constructed a novel molecule, BC-MAP, as a functional immobilizing linker by genetic fusion of MAP with two domains (B and C) of protein A for facile and effective immobilization of antibodies on diverse solid supports with oriented manner without any additional steps and/or pretreatments. A representative scheme of effective immobilization of antibody onto diverse surfaces using functional BC-MAP linker material is shown in FIG. 8.